Early expansion of CD38+ICOS+ GC Tfh in draining lymph nodes during influenza vaccination immune response

T follicular helper (Tfh) cells provide critical help to B cells during the germinal center (GC) reaction to facilitate generation of protective humoral immunity. Accessing the human lymph node (LN) to study the commitment of CD4 T cells to GC Tfh cell differentiation during in vivo vaccine responses is difficult. We used ultrasound guided fine needle biopsy to monitor recall responses in axillary LNs to seasonal influenza vaccination in healthy volunteers. Specific expansion of GC cell subsets occurred exclusively within draining LNs five days postvaccination. Draining LN GC Tfh and precursor-Tfh cells express higher levels of CD38, ICOS, and Ki67, indicating they were significantly more activated, motile, and proliferating, compared to contralateral LN cells. These observations provide insight into the early expansion phase of the human Tfh lineage within LNs during a vaccine induced memory response and highlights early LN immune responses may not be reflected in the periphery

Circulating gluten-specific FOXP3⁺CD39⁺ regulatory T cells have impaired suppressive function in patients with celiac disease

Background Celiac disease is a chronic immune-mediated inflammatory disorder of the gut triggered by dietary gluten. Although the effector T-cell response in patients with celiac disease has been well characterized, the role of regulatory T (Treg) cells in the loss of tolerance to gluten remains poorly understood. Objective We sought to define whether patients with celiac disease have a dysfunction or lack of gluten-specific forkhead box protein 3 (FOXP3)+ Treg cells. Methods Treated patients with celiac disease underwent oral wheat challenge to stimulate recirculation of gluten-specific T cells. Peripheral blood was collected before and after challenge. To comprehensively measure the gluten-specific CD4+ T-cell response, we paired traditional IFN-γ ELISpot with an assay to detect antigen-specific CD4+ T cells that does not rely on tetramers, antigen-stimulated cytokine production, or proliferation but rather on antigen-induced coexpression of CD25 and OX40 (CD134). Results Numbers of circulating gluten-specific Treg cells and effector T cells both increased significantly after oral wheat challenge, peaking at day 6. Surprisingly, we found that approximately 80% of the ex vivo circulating gluten-specific CD4+ T cells were FOXP3+CD39+ Treg cells, which reside within the pool of memory CD4+CD25+CD127lowCD45RO+ Treg cells. Although we observed normal suppressive function in peripheral polyclonal Treg cells from patients with celiac disease, after a short in vitro expansion, the gluten-specific FOXP3+CD39+ Treg cells exhibited significantly reduced suppressive function compared with polyclonal Treg cells. Conclusion This study provides the first estimation of FOXP3+CD39+ Treg cell frequency within circulating gluten-specific CD4+ T cells after oral gluten challenge of patients with celiac disease. FOXP3+CD39+ Treg cells comprised a major proportion of all circulating gluten-specific CD4+ T cells but had impaired suppressive function, indicating that Treg cell dysfunction might be a key contributor to disease pathogenesis

Patients with treated indolent lymphomas immunized with BNT162b2 have reduced anti-spike neutralizing IgG to SARS-CoV-2 variants, but preserved antigen-specific T cell responses

Patients with indolent lymphoma undertaking recurrent or continuous B cell suppression are at risk of severe COVID-19. Patients and healthy controls (HC; N = 13) received two doses of BNT162b2: follicular lymphoma (FL; N = 35) who were treatment naïve (TN; N = 11) or received immunochemotherapy (ICT; N = 23) and Waldenström's macroglobulinemia (WM; N = 37) including TN (N = 9), ICT (N = 14), or treated with Bruton's tyrosine kinase inhibitors (BTKi; N = 12). Anti-spike immunoglobulin G (IgG) was determined by a high-sensitivity flow-cytometric assay, in addition to live-virus neutralization. Antigen-specific T cells were identified by coexpression of CD69/CD137 and CD25/CD134 on T cells. A subgroup (N = 29) were assessed for third mRNA vaccine response, including omicron neutralization. One month after second BNT162b2, median anti-spike IgG mean fluorescence intensity (MFI) in FL ICT patients (9977) was 25-fold lower than TN (245 898) and HC (228 255, p =.0002 for both). Anti-spike IgG correlated with lymphocyte count (r =.63; p =.002), and time from treatment (r =.56; p =.007), on univariate analysis, but only with lymphocyte count on multivariate analysis (p =.03). In the WM cohort, median anti-spike IgG MFI in BTKi patients (39 039) was reduced compared to TN (220 645, p =.0008) and HC (p <.0001). Anti-spike IgG correlated with neutralization of the delta variant (r =.62, p <.0001). Median neutralization titer for WM BTKi (0) was lower than HC (40, p <.0001) for early-clade and delta. All cohorts had functional T cell responses. Median anti-spike IgG decreased 4-fold from second to third dose (p =.004). Only 5 of 29 poor initial responders assessed after third vaccination demonstrated seroconversion and improvement in neutralization activity, including to the omicron variant

A mammalian cell based FACS-panning platform for the selection of HIV-1 envelopes for vaccine development

An increasing number of broadly neutralizing monoclonal antibodies (bnMAb) against the HIV-1 envelope (Env) protein has been discovered recently. Despite this progress, vaccination efforts with the aim to re-elicit bnMAbs that provide protective immunity have failed so far. Herein, we describe the development of a mammalian cell based FACS-panning method in which bnMAbs are used as tools to select surface-exposed envelope variants according to their binding affinity. For that purpose, an HIV-1 derived lentiviral vector was developed to infect HEK293T cells at low multiplicity of infection (MOI) in order to link Env phenotype and genotype. For proof of principle, a gp145 Env model-library was established in which the complete V3 domain was substituted by five strain specific V3 loop sequences with known binding affinities to nMAb 447-52D, respectively. Env genes were recovered from selected cells by PCR, subcloned into a lentiviral vector (i) to determine and quantify the enrichment nMAb binders and (ii) to generate a new batch of transduction competent particles. After 2 selection cycles the Env variant with highest affinity was enriched 20-fold and represented 80% of the remaining Env population. Exploiting the recently described bnMAbs, this procedure might prove useful in selecting Env proteins from large Env libraries with the potential to elicit bnMAbs when used as vaccine candidates

Circulating gluten-specific, but not CMV-specific, CD39(+) regulatory T cells have an oligoclonal TCR repertoire

Objectives: Understanding the T cell receptor (TCR) repertoire of regulatory CD4+ T-cell (Treg) populations is important for strategies aiming to re-establish tolerance in autoimmune diseases. We studied circulating deamidated gluten-epitope-specific CD39+ Tregs in patients with coeliac disease following an oral gluten challenge, and we used cytomegalovirus (CMV)-specific CD39+ Tregs from healthy controls as a comparator population. Methods: We used the OX40 assay to isolate antigen-specific Tregs by induced surface co-expression of CD25, OX40 and CD39. RACE PCR amplification and Sanger sequencing of the TCR β chain were used to analyse repertoire diversity. Results: We found that, following oral gluten challenge, circulating gluten-specific CD39+ Tregs had an oligoclonal TCR repertoire that contained public clonotypes. Conversely, the TCR repertoire of CMV-epitope-specific CD39+ Tregs from healthy controls was polyclonal. Discussion: These data indicate that a biased TCR repertoire is not inherent to CD39+ Tregs, and, in this case, is apparently driven by the HLA-DQ2.5-restricted deamidated gluten peptide in coeliac disease patients. Conclusion: This is the first assessment of the TCR repertoire within circulating human Tregs specific for foreign antigen. These data enhance our understanding of antigen-specific CD4+ responses in the settings of chronic inflammation and infection and may help guide immunomonitoring strategies for CD4+ T cell-based therapies, particularly for coeliac disease

Maintenance of functional CD57+cytolytic CD4+T cells in HIV plus elite controllers

Cytolytic CD4+ T cells play a prominent role in chronic viral infection. CD4+ CTLs clones specific for HIV-1 Nef and Gag are capable of killing HIV-1 infected CD4+ T cells and macrophages. Additionally, HIV-specific cytolytic CD4+ T cell responses in acute HIV infection are predictive of disease progression. CD57 expression on CD4s identifies cytolytic cells. These cells were dramatically increased in chronic HIV infection. CD57 expression correlated with cytolytic granules, granzyme B and perforin expression. They express lower CCR5 compared to CD57– cells, have less HIV total DNA, and were a minor component of the HIV reservoir. A small percentage of CD57+ CD4+ CTLs from EC were HIV-specific, could upregulate IFNγ with Gag peptide stimulation, express cytolytic granule markers and maintain TbethighEomes+ transcription factor phenotype. This was not observed in viraemic controllers. The maintenance of HIV-specific CD4 cytolytic function in Elite controllers together with CD8 CTLs may be important for the control of HIV viraemia and of potential relevance to cure strategies

Human antigen-specific CD4⁺CD25⁺CD134⁺CD39⁺ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

Human Ag-specific CD4+ T cells can be detected by their dual expression of CD134 (OX40) and CD25 after a 44 hours stimulation with cognate Ag. We show that surface expression of CD39 on Ag-specific cells consistently identifies a substantial population of CD4+CD25+CD134+CD39+ T cells that have a Treg-cell-like phenotype and mostly originate from bulk memory CD4+CD45RO+CD127lowCD25highCD39+ Treg cells. Viable, Ag-specific CD25+CD134+CD39+ T cells could be expanded in vitro as cell lines and clones, and retained high Forkhead Box Protein 3, CTLA-4 and CD39 expression, suppressive activity and Ag specificity. We also utilised this combination of cell surface markers to measure HIV-Gag responses in HIV+ patients before and after anti-retroviral therapy (ART). Interestingly, we found that the percentage of CD39- cells within baseline CD4+ T-cell responses to HIV-Gag was negatively correlated with HIV viral load pre-ART and positively correlated with CD4+ T-cell recovery over 96 weeks of ART. Collectively, our data show that Ag-specific CD4+CD25+CD134+CD39+ T cells are highly enriched for Treg cells, form a large component of recall responses and maintain a Treg-cell-like phenotype upon in vitro expansion. Identification and isolation of these cells enables the role of Treg cells in memory responses to be further defined and provides a development pathway for novel therapeutics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Evaluation of commercially available viral transport medium (VTM) for SARS-CoV-2 inactivation and use in point-of-care (POC) testing

Critical to facilitating SARS-CoV-2 point-of-care (POC) testing is assurance that viruses present in specimens are inactivated onsite prior to processing. Here, we conducted experiments to determine the virucidal activity of commercially available Viral Transport Mediums (VTMs) to inactivate SARS-CoV-2. Independent testing methods for viral inactivation testing were applied, including a previously described World Health Organization (WHO) protocol, in addition to a buffer exchange method where the virus is physically separated from the VTM post exposure. The latter method enables sensitive detection of viral viability at higher viral titre when incubated with VTM. We demonstrate that VTM formulations, Primestore® Molecular Transport Medium (MTM) and COPAN eNAT™ completely inactivate high-titre SARS-CoV-2 virus (>1 × 107 copies/mL) and are compatible with POC processing. Furthermore, full viral inactivation was rapidly achieved in as little as 2 min of VTM exposure. We conclude that adding certain VTM formulations as a first step post specimen collection will render SARS-CoV-2 non-infectious for transport, or for further in-field POC molecular testing using rapid turnaround GeneXpert platforms or equivalent

The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype

Background Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. Methods We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. Results A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. Conclusions We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV